This research is focused on mechanisms to enhance the cytolytic response to HIV-1 antigens, with the goal of developing a clinically effective vaccine against HIV-1. Two novel advances underlie the application of this research to HIV-1. (1) A strategy has been developed for targeting gene products of DNA vaccines to a cellular trafficking pattern that accesses the major histocompatibility class II (MHC II) pathway for processing and presentation of antigenic epitopes to the helper T cell arm of the immune response mechanism. This targeting of antigen has been shown to result in a marked enhancement of the cytolytic immune response to the antigen. (2) A non-viral system for in vivo delivery of genes as a DNA-gelatin nanosphere coacervate is being applied for the delivery of the DNA vaccine to antigen-presenting cells, and for the co-delivery of cytokines that augment the Th1 CD4+ subset of helper T cells so as to potentiate the cytolytic T cell response of the immune system. The principle goals of the proposed research are to perfect these systems with the HIV-1 gag protein as the initial antigen, and to prove the feasibility of these approaches so that they may be considered for human clinical trials. The research design is, first, to prepare DNA expression plasmid constructs encoding a chimeric gag protein containing the signal, transmembrane and cytoplasmic targeting sequences of the LAMP lysosomal membrane protein, and to test mice vaccinated with the plasmid as naked DNA for the expected enhanced presentation of the antigen to CD4+ helper T cells and augmented cytolytic T cell response. The experiments also will test the effect on the immune response of modifications of the gag gene required for nuclear transport of the mRNA in the absence of the rev gene: gag gene constructs containing mutations in the INS inhibitory instability elements, the simian retrovirus type 1 constitutive transport element (CTE), or a combination of the two. Next, after selection of the appropriate plasmid construct, the DNA will be incorporated into nanospheres to study the cytolytic response of the mouse vaccinated with nanospheres as compared to naked DNA, and to perfect the nanosphere formulation for this application as a vaccine. Finally, the appropriate formulation will be tested for the co-delivery with gag DNA of cytokines, especially IL-2, IL-12, gamma-INF and GMCSF, in the augmentation of the cytolytic response.